Model systems are used to understand how neurotransmission is regulated and how it malfunctions during diseases. Drosophila, the fruit fly, is an attractive model organism because it is easy to genetically alter, fast to breed, inexpensive, and has complex behaviors. Drosophila has many neurotransmitter systems similar to mammals, including dopaminergic and serotonergic systems. While dopamine and serotonin measurements have been made in the larval Drosophila ventral nerve cord, no measurements of endogenous release have been made in the adult fly brain. Adult flies have a fully developed nervous system and would be a good model system for studying the regulation of neurotransmitters in mental health diseases. The long-term goal of my lab is to develop new analytical tools to understand real-time neurotransmission in biological systems. The goal of this project is to characterize the regulation of dopamine and serotonin signaling in specific neuropil of the adult and larval Drosophila. Specifically, we will determine which serotonin receptors act as auto receptors and how dopamine is regulated by RING finger protein 11 (RNF11), a modulator of the NF-?B pathway. The central hypothesis is that neurotransmitter regulation varies with age and by neuropil. Aim 1 is to compare dopamine release in discrete areas of the larval and adult CNS. Release will be evoked by optically activating genetically-inserted CsChrimson and the effect of aging on dopamine release will be studied in the adult. In Aim 2, we will explore the regulation of dopamine by RNF11 using RNAi knockdown of RNF11. The effect of D2 auto receptor antagonist antipsychotics will be studied in RNF11 RNAi flies, to investigate the extent to which RNF11 affects auto receptor regulation of dopamine. These will be the first studies of the effect of RNF11 on dopamine signaling in any species. In Aim 3, we will investigate stimulated serotonin release and reuptake in larvae (protocerebrum and VNC) and adults (mushroom bodies, VNC). The serotonin system is a target of many treatments for mental illness, so understanding it in flies will enable its use as a model organism of mental diseases. Finally, for Aim 4, we will determine the effect of serotonin auto receptors on serotonin regulation in larvae and adults using both pharmacological and genetic approaches. The expected outcomes of this work are better methods for characterizing neurotransmission in specific Drosophila neuropil and an understanding of the regulation of dopamine and serotonin release throughout Drosophila development. The research is significant because it will provide foundational knowledge of neurotransmitter signaling necessary to use the powerful genetic tools available for Drosophila to study the effects of specific genes in controlling neurotransmission. Future studies could examine how dopaminergic and serotonergic neurotransmission control behaviors or function in diseases in adult Drosophila.